Research Project 1 (RP1) builds on the progress and success of Projects 1 and 2 in ICMIC-2. Our hypothesis is that both constitutive and PSMA-activated reporter gene imaging of PSMA directed T cell targeting, distribution and persistence can be performed concurrently in patients with castrate-resistant prostate cancer. The central theme of this proposal is consistent with our ICMIC: cancer biology integrated with real-time, noninvasive imaging for application in individual patient care and management'. A phase 1 clinical study will determine whether genetically altered, PSMA-directed, adoptive T cell therapy in human subjects results in adequate T cell activation as well as targeting and persistence in prostate cancer (Aim 1). These are novel imaging experiments involving dual human reporter genes, not previously performed in human subjects with cancer. The imaging results will be compared to standard clinical treatment-response measures. In parallel, we will also explore in animal models whether an acidic tumor microenvironment and high lactate levels adversely effect T cell targeting, activation and persistence using a unique combination of imaging strategies (Aim 2). We will also determine in animal models of prostate cancer whether modulation of PSMA expression can enhance PSMA-directed T cell therapy using our novel reporter systems (Aim 3). If positive results in Aims 2 and 3 are obtained, similar studies could be readily performed in patients. Our long-term goals are to further develop unique clinical trials of adoptive therapy that includes reporter imaging to track and monitor T cell activation and persistence in patients. We expect to be able to address novel questions in clinical immunology and immunotherapy, including the effects of the tumor microenvironment and modulation of CAR-directed tumor antigens on treatment response. The relevance and impact of these studies are that: 1) imaging the trafficking and activation status of PSMA directed T cells will provide substantial added value to the management of prostate cancer patients undergoing adoptive T cell therapy; 2) the imaging strategies developed and validated in this project could be readily applied to other cancers, as well as applied in other adoptive therapy (e.g., stem cell) protocols and nonmalignant disease; 3) the effect of an acidic-high lactate-hypoxic microenvironment could have a significant negative impact on T cell targeting and activation. These hypotheses will initially be explored in animal models. Importantly, the assay systems, imaging strategies and therapeutic counter-measures could be applied and tested in future patient studies, and could serve as a model for similar studies in other cancers as well as for adoptive stem cell therapy in other diseases.